Salmonella cause 1.4 million cases of gastroenteritis and enteric fever per year in the US and lead all other foodborne bacterial pathogens as a cause of death. The most serious disease results from S. typhimurium survival in phagocytes, which normally kill bacteria by producing a variety of antimicrobials including superoxide (O2-). The mechanism by which phagocytic 02- damages bacterial cells is completely unknown. S. typhimurium strain 14028 possesses two periplasmic superoxide dismutases. SodCI, encoded on the Gifsy-2 phage, contributes significantly to virulence by protecting against phagocytic 02-. The chromosomally encoded SodCII, which we have shown is expressed during infection, does not contribute to survival, even in the absence of SodCI. The two enzymes behave similarly in vitro with the notable exception that SodCI is not released by osmotic shock, a phenomenon we term "tethering." This is a novel property for a periplasmic protein. We hypothesize that SodCII is produced but is non-functional, probably due to proteolytic degradation in the macrophage phagosome, and that the physical or spatial association of SodCI with some periplasmic component accounts for its preferential role in virulence. The specific aims of this proposal are to: 1. Determine the fate of SodCI and SodCII in the phagosome. We will simultaneously monitor the production of both SodCI and SodCII protein during infection in an animal as well as in tissue culture macrophages. 2. Determine the structural and functional characteristics of SodCI that allow it to protect against phagocytic superoxide. We will exploit the differential activity of SodCI and SodCII. Hybrid proteins will be constructed and characterized. SodCs from other pathogenic bacteria and specific site directed mutants will also be tested for the ability to complement SodCI. The ability to combat phagocytic superoxide will be correlated with other characteristics including release by osmotic shock. 3. Determine if tethering to the periplasm is required for function in vivo and understand the biochemical nature of tethering. A genetic screen for mutants that are no longer tethered as well as a biochemical identification of what SodCI interacts with in the periplasm will lead to an understanding of tethering and its role in SodCI function. This research addresses a fundamental issue in innate immunity and has implications for combating a variety of important pathogens.